title: Purine nucleoside phosphorylase targeted by annexin v to breast cancer vasculature for enzyme prodrug therapy.
abstract: OBJECTIVE
  The targeting of therapeutics is a promising approach for the development of new cancer treatments that seek to reduce the devastating side effects caused by the systemic administration of current drugs. This study evaluates a fusion protein developed as an enzyme prodrug therapy targeted to the tumor vasculature. Cytotoxicity would be localized to the site of the tumor using a protein fusion of purine nucleoside phosphorylase (PNP) and annexin V. Annexin V acts as the tumor-targeting component of the fusion protein as it has been shown to bind to phosphatidylserine expressed externally on cancer cells and the endothelial cells of the tumor vasculature, but not normal vascular endothelial cells. The enzymatic component of the fusion, PNP, converts the FDA-approved cancer therapeutic, fludarabine, into a more cytotoxic form. The purpose of this study is to determine if this system has a good potential as a targeted therapy for breast cancer.
  
  
  METHODS
  A fusion of E. coli purine nucleoside phosphorylase and human annexin V was produced in E. coli and purified. Using human breast cancer cell lines MCF-7 and MDA-MB-231 and non-confluent human endothelial cells grown in vitro, the binding strength of the fusion protein and the cytotoxicity of the enzyme prodrug system were determined. Endothelial cells that are not confluent expose phosphatidylserine and therefore mimic the tumor vasculature.
  
  
  RESULTS
  The purified recombinant fusion protein had good enzymatic activity and strong binding to the three cell lines. There was significant cell killing (p<0.001) by the enzyme prodrug treatment for all three cell lines, with greater than 80% cytotoxicity obtained after 6 days of treatment.
  
  
  CONCLUSIONS
  These results suggest that this treatment could be useful as a targeted therapy for breast cancer.
authors.affiliation: Bioengineering Center and the School of Chemical, Biological and Materials Engineering, University of Oklahoma, Norman, Oklahoma, United States of America.
authors.forename: John J
authors.initials: JJ
authors.lastname: Krais
authors.forename: Olivier
authors.initials: O
authors.lastname: De Crescenzo
authors.forename: Roger G
authors.initials: RG
authors.lastname: Harrison
mesh_terms: D017304:Annexin A5 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000970:Antineoplastic Agents / Q000008:administration & dosage / Q000494:pharmacology* / Q000633:toxicity; D001943:Breast Neoplasms / Q000188:drug therapy / Q000235:genetics / Q000378:metabolism* / Q000473:pathology*; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000378:metabolism; D005260:Female; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D058990:Molecular Targeted Therapy; D009389:Neovascularization, Pathologic / Q000235:genetics / Q000378:metabolism*; D011355:Prodrugs / Q000008:administration & dosage / Q000494:pharmacology* / Q000633:toxicity; D011485:Protein Binding; D011487:Protein Conformation; D055550:Protein Stability; D011683:Purine-Nucleoside Phosphorylase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D017304:Annexin A5; D000970:Antineoplastic Agents; D011355:Prodrugs; D011993:Recombinant Fusion Proteins; D011683:Purine-Nucleoside Phosphorylase